Project Summary The goal of cell division is to reliably produce two identical daughter cells, each with an exact copy of the original genetic material. When this process goes awry, a common result is aneuploidy, which is a leading cause of birth defects and a key characteristic of cancer. Mitotic cell division critically depends on kinetochores, structures that orchestrate chromosome segregation and integrate all aspects of the mitotic machinery to ensure mitosis is executed with high fidelity. Kinetochores physically connect chromosomes to spindle microtubules (MTs), and they regulate the strength of these connections so that erroneously-attached MTs are released, and correctly-attached MTs are stabilized. Importantly, kinetochores ensure that cells do not exit mitosis if chromosomes fail to attach, or are incorrectly attached, to MTs. Much progress has been made in identifying the kinetochore proteins that participate in these processes; however, how these molecules function in concert to ensure the accuracy of chromosome attachment and segregation, and to ensure timely mitotic progression remains poorly understood. Our lab has been instrumental in defining how kinetochores regulate MT attachment, and how this fundamental aspect of mitosis is integrated into controlling cell cycle progression. Additionally, our lab has begun to make significant inroads to understanding how oncogenic transformation leads to deregulated kinetochore function, and how these defects lead to cancer cell-specific vulnerabilities that can potentially be exploited for cancer therapies. Our expertise in studying kinetochore function in combination with our newly developed experimental approaches ? especially those that provide increased spatial and temporal resolution of kinetochore proteins ? puts us in a strategic position to resolve how kinetochores ensure accurate chromosome segregation and drive mitotic progression. In the next five years, our research will focus on four areas. We will: (1) Examine the mechanisms of kinetochore-MT attachment regulation using biochemical and cell biological tools, as well as new assays to track the dynamics of specific phosphorylation events in cells with high temporal resolution; (2) Investigate how Aurora B kinase, the ?master? regulator of attachment, is recruited to discrete centromere and kinetochore regions with precise temporal control using in-cell mutagenesis approaches, proximity-dependent interaction/mass spectrometry analysis, and phospho-modification tracking techniques; (3) Probe how kinetochore-MT attachment status is communicated to the spindle assembly checkpoint, in part by using super-resolution imaging to map the changes in kinetochore architecture that occur upon stable MT attachment; (4) Determine how oncogenic signaling leads to kinetochore-MT attachment deregulation using a tumor progression model system built from primary cells. In sum, our studies will provide critical insight into the fundamental mechanisms that regulate kinetochore-MT attachment, and that integrate this critical mitotic function with other mitotic processes including chromosome architecture, spindle MT dynamics, and checkpoint signaling. !